Lyme disease is the most common vector-borne disease in the United States. Understanding the molecular mechanisms by which Borrelia burgdorferi, the etiologic agent, survives in nature will help drive development of innovative strategies for disease prevention. We have demonstrated that the function(s) of Borrelia iron- and copper-binding protein A (BicA), which is encoded by chromosomal gene bb0690, is critically important for B. burgdorferi survival in an infectious life cycle through the tick and te mammal. We have discovered that iron and copper are present in B. burgdorferi and their homeostasis are altered in a mutant deficient in BicA and in a mutant deficient in a metal transporter. To date, all evidence either for or against the presence of iron in B. burgdorferi is based on in vitro experiments where the spirochetes were grown in artificial media. Therefore, a critical question to be addressed is whether iron and copper play any role in the natural life cycl of this tick-borne pathogen. This is the question we are seeking to address with the experiments outlined in this proposal. Iron and copper are important metal cofactors in many biological reactions, but iron and copper can be toxic to cells due to their abilities to catalyze the Fenton r Fenton-like reaction. Hence, organisms utilizing iron or copper have evolved many strategies to control intracellular levels of free iron and copper. Metal-binding proteins such as ferritins and metallothioneins represent one such strategy. Our preliminary data show that (i) BicA is a novel fusion between an iron-binding ferritin-like molecule and a copper-binding metallothionein; (ii) B. burgdorferi lacking BicA has reduced levels of iron and copper, and is more sensitive to iron and copper toxicity; and (iii) B. burgdorferi requires BicA for full infectivity in mice as well as persistence in the tick and subsequent transmission to a new host. Thus, we hypothesize that iron and copper detoxification by BicA is critical to B. burgdorferi survival in the tick and the mammal. To test this hypothesis, we propose to demonstrate that (i) iron and copper are the metals bound by native BicA from B. burgdorferi; (ii) iron- and copper-binding by BicA protects B. burgdorferi from iron and copper toxicity, respectively; (iii) the iron- and copper-binding activities of BicA are important for B. burgdorferi survival in its infectious life cycle through te tick and the mammal. Successful execution of these lines of investigation will not only elucidate molecular mechanisms underlying B. burgdorferi persistence in nature but also help advance our understanding of metal biology in this important human pathogen.